INTRODUCTION:

Eye on chip is also known as organ-on-a-chip (OOC), microphysiological system, or tissue-on-a-chip. OOCs become a powerful tool in medicine and healthcare with organisms cultured on ex vivo microfluidic chips to perform specific functions of the body or multiple organs.¹ The first OOC model was created by combining a lung cell culture model with a heart machine to evaluate the effects and toxicity of drugs in vitro.² OOCs are designed to model organotypic cellular structure and function, biochemical factors, and biophysical cues at a small scale; this may be useful in disease models and drug testing. Many types of organ devices have been developed and used for biological and medical research, such as liver chips, kidney chips, heart chips, eye chips, and blood-brain barrier chips.^2,3

The manufacturing materials of organ chips does not disrupt cellular microenvironment components and maintain fluid stability. The most commonly used material for OOC is polydimethylsiloxane (PDMS).^4,5

The development of the Eye-on-Chip (EOC) model is currently being worked on. These EOCs help influence eye physiology by analyzing the pharmacokinetics (PK) and pharmacodynamics (PD) of drugs using a single platform.² Technology could become a way to develop personalized treatments by using the patients’ own cells to achieve personalized medicine approaches.

Eye on a chip:

The National Eye Institute (NEI) at the U.S. National Institutes of Health (NIH) support’s research that helps prevent and treat eye diseases and other disorders of vision.⁶ EOC has greatly aided the diagnosis, treatment, and research of various ophthalmological disorders. Dry eye syndrome is an eye disease that caused by multiple factors mainly imbalances in tear film homeostasis accompanied by tear film instability and ocular surface damage. To understand the pathophysiology of DED, progress has been made in reconstructing the physiological and pathological ocular models, aiming to recapitulate key aspects in the ocular development and diseases.⁷

Developments are also underway for corneal tissue chips that could be used in preclinical drugs evaluations. The development of EOC models that simulate blinking by combining tears with 3D printed eyelids will impact the development of medicine.⁶

How is eye on a chip better than other Ophthalmic Models for DED?

Researchers have been examining the in vitro and in vivo modelling of various components separately, such as the conjunctival epithelium and lacrimal glands, in order to understand dry eyes disease by taking into account the ocular surface and tear film. Several models have been proposed for this purpose.⁸

A. In vivo animal models:

Animal models of dry eye are crucial for drug discovery and development. They have been used to investigate the aetiology, diagnosis, and treatment of several eye conditions.⁹ Unfortunately, due to interspecies variability, animal research is not always precise during clinical trials and is constrained by expensive expenses and ethical concerns.¹⁰ In the rabbit lacrimal gland, for instance, there are mixed populations of acini that produce mucous and serous, in contrast to the predominance of serous acini in the human lacrimal gland. Other glands found in rabbit eyes, such as the Harderian gland and the nictitating membrane (NM), also contribute to the tear film. While designing an animal model of DED or carrying out studies, the sex-related variations in the lacrimal gland and ocular surface morphology should also be taken into account.^11,12

B. Ex vivo explant cultures:

Explant cultures play an important role in ophthalmic research, and plant culture is mainly used for the culture of retina and corneal tissue from animal or human donors. An organ or a tiny tissue slice that may be surgically removed and kept in culture is referred to as a transplant. Although human tissue is preferred over animal tissue in transplant cultures, both will get over the problem of interspecies variances.¹³ On the other hand, this ex vivo cultures often encounter significant challenges in maintaining viability and functionality over extended periods of time.¹⁰

Advantages of eye on a chip:

EOC models combine the benefits of every type mentioned above. Compared to traditional cell assays or animal models, these models are expected to be more accurate and reliable at anticipating how human cells and organs would react to substances, poisons, or treatment strategies.^14,15 Because EOC technology can minimize or replace animal studies, its actual socio-economic effects extend well beyond this industry area. EOCs can be employed effectively in the investigation of dry eyes disease because they can integrate multi-cell type 3D tissues with physiological structure and function, which has the potential to overcome many of these constraints.¹⁰

Dry eye disease:

A multifactorial disease of the tears and ocular surface, dry eye disease is characterized by symptoms of pain, visual impairment, and instability of the tears' film, which can lead to damage to the ocular surface. It is associated with subacute ocular surface inflammation and elevated tear film osmolarity.

DED can be categorized into two groups on the basis of pathophysiology.¹⁶

1. Dry eye with reduced tear production (aqueous deficient)

2. Dry eye with increased evaporation of the tear film known as the hyper evaporative type

Epidemiology: Between 5% and 34% of people worldwide are suffering from dry eyes. As one ages, the occurrence rises noticeably. The substantial variation in prevalence in the figures can be attributed to methodological variances, regional disparities, and changes in the study populations. Functional vision is impaired by dry eye, particularly when reading, using a computer, or driving. Environmental factors that contribute to the occurrence of DED include high winds, low humidity levels, air-conditioned, and other similar circumstances.^16,17 Various risk factors are given in Fig. 1:

Fig. 1: Risk factors for DED.^15,18

Pathophysiology:

DED is primarily caused by a disruption in the homeostasis of the ocular surface and tear film. Tear film instability, hyperosmolarity, inflammation, and ocular surface injury are possible causes of this disturbance.¹⁷ Numerous investigations have demonstrated that desiccation is a strong stressor on the surface of the eyes that can set off a secondary immune response and create a vicious cycle Fig. 2. Hyperosmolarity causes apoptosis and activates mitogen-activated protein kinases (MAPKs), chemokines, matrix metalloproteinases (MMP) like MMP-3 and MMP-9, and pro-inflammatory cytokines including IL-1β, TNF-α, and IL-6. It has been demonstrated that the intricate interactions between these inflammatory mediators cause them to upregulate one another, intensifying the inflammatory cascade that eventually results in dry eye disease.¹⁷ Fig. 2:

Fig. 2: Showing pathophysiology of DED

Characteristics of dry eyes

A human eye's typical blink rate ranges from 15.5 ± 13.7 blinks per minute. However, when reading or using a computer, the blink rate is greatly decreased to 5.3 ± 4.5 blinks/minute, which encourages the evaporation of tear fluid. Patients with dry eye typically experience incomplete blinking and a decreased blink interval of roughly 6 seconds to 2.6 seconds.¹⁸

It is also found that tear film meniscus height was 0.2 ± 0.09 mm in patients with dry eye versus 0.5 ± 0.02 mm in individual with healthy eyes. In clinical practice, a tear film meniscus below 0.2 mm is regarded as pathological.¹⁸

The stability of tear film is described by tear film break-up time (TFBUT). The normal range of TFBUT lies between 20 and 30 seconds. Values below 10 seconds are definitely pathological.¹⁸

Techniques utilizing organ chip in DED study:

A lot of attempts have been made to study DED but in this article we will study about three techniques that can be employed in order to study the disease morphology and also for drug development via use of organ chips. These three techniques are following;

1. Cornea on a chip

2. Human blinking eye on a chip

3. Lacrimal gland on a chip

1. Cornea on a chip:

Puleo et al. made the first attempt to culture cornea cells in microfluidic devices in 2009. They succeeded in developing a bilayer structure containing a corneal epithelial layer and a layer of stromal cells on a collagen vitrigel (CV) substrate.¹⁹ Further a vacuum network was applied to both layers to seal the CV between two opposing microchannels, which helps to rehydrate parts of the CV to achieve a thin and transparent gel membrane. This vacuum method provides the advantage of removing the membrane easily out of the chip for study purpose.²⁰ The authors employed the system to screen potential eye irritants by exposing it to NaOH, which reduced the tissue integrity drastically. The model was then used to measure transepithelial permeability.¹⁹

Limitations: Cornea chips have been used to study pharmacokinetics and preclinical drug evaluation. Bennet et al. performed a drug study on their model with Pred Forte (Prednisolone 1%) and Zaditor (Ketotifen) to assess the functionality of their model in drug permeability.²¹ They found that the pulsatile tear flow had the most similarity to the human eye than the continuous flow or static condition. They found that physiology and permeability rate of static EOC is very different from the normal eye. Normal eye contains dynamic conditions such as blinking eye lid.¹⁹ A further advanced cornea-on-a-chip, model was much needed and then a ‘human blinking eye-on-a-chip’ system was presented. As an improvement over the cornea-only chip, this model aimed to mimic the entire ocular surface structure, including a recapitulation of tear-film dynamics and spontaneous eye blinking in humans.¹⁰

2. Human blinking eye on a chip:

The characteristics of static eye on a chip model are far away different from the characteristics of actual eye. It has been found that the human eyelids are not merely for the purpose of protecting eyes. An important function of these protective eyelids is to maintain the integrity of corneal tear film. Increasing blink amplitude thickens the lipid layer that overlies the aqueous layer of the tear film. This thickening reduces evaporation of the aqueous layer. Because each blink reforms the tear film, increasing blink frequency reduces tear film break-up.²²

So, considering the importance of blinking effect of human eye various models of blinking eye are given. A recent study by Bennet et al. introduced a new corneal epithelium-on-a-chip model which can mimic the blink characteristics similar to the human eye. The purpose of this model was to mimic the tear flow associated with eye-blinking as this feature is crucial for recapitulating the physiological microenvironment in mass transport studies of ocular drugs because tear flow and blinking are the two primary parameters influencing drug residence time on the cornea.¹⁰A significant breakthrough in the development of an ocular surface chip was the chip development by Seo et al.¹⁹

2.1 Imitating eye blinking and physiological tear film dynamics.

In order to incorporate the blinking eyelid like structure a biomimetic analog of eyelids made out of biocompatible hydrogel is used. The motion of the eyelid was controlled by an electromechanical actuator to generate cyclic back-and-forth movements at 0.2 Hz following the kinematics of spontaneous blinking in the normal human eye. Tear production and secretion from the lacrimal glands were simulated by controlled outflow of artificial tears through small orifices in the tear channel at physiological rates of tear secretion in the human eye. The secreted fluid was then pushed by the eyelid during its downward movement and spread over the scaffold to form a liquid film. This actuation also resulted in the flow of excess tears into the drainage channel.²³ This model ensures the maintenance of a very thin and uniform tear film of approximately 6 µm thickness which is within the range of in vivo values.¹⁹

2.2 Application of human blinking EOC:

This EOC model includes a blinking corneal surface and can be used to induce a DED phenotype.²⁴ By reducing the frequencies of blinking from 12 to 6 times per minute and adjusting the humidity of the environment allows for the simulation of an evaporative DED model. Importantly, this EOC model also simulates the cellular changes similar to those observed in DED in vivo. For example, inflammatory cytokines such as interleukin IL-1ß, TNF-a, IL-8 and matrix metalloproteinase MMP-9 were over expressed upon the reduction of blinking frequency in the chip.

Jeongyun Seo et al. assessed IL-8, TNF-α, IL-1β, and MMP-9 expression in their model after inducing DED and monitored the response of these cytokines to DED treatment by administering lubricin Fig. 3.²⁴ The DED model showed a marked decrease in inflammatory markers upon lubricin administration, in agreement with the findings of clinical trials. Hence this EOC model can be used to compare the concentration of these inflammatory mediators in diseased condition and the normal eye.²⁴

Fig. 3: Showing the concentrations of inflammatory mediators (IL-8, MMP-9,TNF-α and IL-1β) in the normal and the DED groups plotted against the duration of culture.²⁵

3. Lacrimal gland on a chip:

The lacrimal glands (LGs) secrete tear fluids, which contain water, electrolytes, and various secreted substances to the ocular surface. Tears play physiologically important roles in maintaining the homeostatic environment of the ocular surface epithelium, such as lid lubrication, hydration, antimicrobial activity, and protection of the ocular surface epithelium. The shortage of tears from LGs, which is induced by aging and various pathological conditions, causes DED.¹⁹ It has been found that DED occurs twice as often in women than in men.²⁶ This variation occurs because of significant, sex-associated differences in the structure and function of the lacrimal gland.²⁷

A primary cause for severe DED is lacrimal gland (LG) insufficiency, which may occur due to aging, trauma, radiation therapy, or autoimmune diseases and leads to a failure of lacrimal tear secretion. In severe cases, complete destruction of the LG tissue occurs and current therapies will neither alleviate the patient’s symptoms nor restore lacrimal function. Therefore, engineering of secretory active LG tissue emerges as a promising goal.²⁸

An in vitro model of ocular surface and tear film has been developed by various researchers to study the pathophysiology of lacrimal glands on spheroids which later on can be used to develop chips. However, combining these lacrimal gland cell types with other parts of ocular surface to create a functional tear film has not been studied. In this model all components of the ocular surface are closely integrated together, and one component can have substantial influence over the secretory function of other. Inflammation was induced to mimic DED in the coculture model system, and its response to therapeutics was compared to monocultures.⁸

DISCUSSION:

1. Regulatory authorities and protocols:

OOC are at their starting stage and are available in market their marketing and utilization by pharma companies requires approval by regulatory bodies. Till now organ chip technology has not been specifically classified by any major regulatory body such as the United States Food and Drug Administration (U.S. FDA), the European Medicines Agency, and/or the Medicines and Healthcare Products Regulatory Agency in the United Kingdom. However, it has been found that most developers of organ on a chip technology follow one of the following three guidelines: ISO 9001:2015, FDA 21 CFR Part 58, and FDA FD&C Act Section 507. Regulatory agencies should also lay guidelines and complete process for the validation of organ on a chip technology for variety of potential applications including disease model development. In order to hasten the screening process of organ chips there is a need to transform organ chip technology into high throughput organ on a chip technology.²⁹

2. General requirements for approval:

In order to maintain the quality of OOC and compliance with regulatory requirements, companies usually apply good manufacturing practice (GMP). The materials used for OOC needs to be biocompatible and should not interfere with the scientific result for this purpose PDMS is most widely used for manufacturing microfluidic devices. Also, OOC devices are rarely completely standalone and thus need to be compatible with other laboratory equipment. To assess the accuracy of biological performance of OOC devices, it is necessary to identify relevant biomarkers and assays and such demonstration usually involves comparison with other in vitro cultures, animal data from various species, and human data when available.³⁰

CONCLUSION:

Dry eye is emerging problem and is affecting the life style of large population; a lot of attempts are done to study the pathophysiology of disease. Ophthalmic chips will inevitability change the way we do in vitro eye research in the future and can be used as alternative model for DED. These chips also provide us with the possibility to study individualized differences in disease manifestations and hence can be used to provide the treatment based on individual symptoms.

Chips are mainly used in clinical trials to decrease the dependence on animals for drug discovery and also by the use of these chips overall cost of drug discovery can be decreased. Many times, during drug discovery new lead molecules pass the preclinical trial stage but fails during the human trial stage due to interspecies variation. This results into unnecessary wastage of money and resources. Chips can be used for disease modeling of dry eyes disease but in order to make the chips work with their full potential lot of work is required to be done. The major limitation of these chips is that interlinking of various parts of eyes and body is required in order to study the effect of one part on the activity of the other.

CONFLICT OF INTEREST:

The authors have no conflicts of interest regarding this investigation.

REFERENCES:

1. Yang Q, Lian Q, Xu F. Perspective: Fabrication of integrated organ-on-a-chip via bioprinting. Biomicrofluidics. 2017; 11(3). doi:10.1063/1.4982945

2. Ma C, Peng Y, Li H, Chen W. Organ-on-a-Chip: A New Paradigm for Drug Development.

3. Duffy DC, McDonald JC, Schueller OJA, Whitesides GM. Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal Chem. 1998; 70(23): 4974-4984. doi:10.1021/ac980656z

4. Danku AE, Dulf EH, Braicu C, Jurj A, Berindan-Neagoe I. Organ-On-A-Chip: A Survey of Technical Results and Problems. Front Bioeng Biotechnol. 2022; 10. doi:10.3389/fbioe.2022.840674

5. Bhatia SN, Ingber DE. Microfluidic organs-on-chips. Nat Biotechnol. 2014; 32(8): 760-772. doi:10.1038/nbt.2989

6. Wright CB, Becker SM, Low LA, Tagle DA, Sieving PA. Improved Ocular Tissue Models and Eye-On-A-Chip Technologies Will Facilitate Ophthalmic Drug Development. Journal of Ocular Pharmacology and Therapeutics. 2020; 36(1): 25-29. doi:10.1089/jop.2018.0139

7. Bai J, Wang C. Organoids and Microphysiological Systems: New Tools for Ophthalmic Drug Discovery. Front Pharmacol. 2020; 11. doi:10.3389/fphar.2020.00407

8. Lu Q, Yin H, Grant MP, Elisseeff JH. An in Vitro Model for the Ocular Surface and Tear Film System. Sci Rep. 2017; 7(1). doi:10.1038/s41598-017-06369-8

9. Spöler F, Frentz M, Schrage NF. Towards a New in Vitro Model of Dry Eye:The Ex Vivo Eye Irritation Test. Vol 45.; 2010.

10. Haderspeck JC, Chuchuy J, Kustermann S, Liebau S, Loskill P. Organ-on-a-chip technologies that can transform ophthalmic drug discovery and disease modeling. Expert Opin Drug Discov. 2019; 14(1): 47-57. doi:10.1080/17460441.2019.1551873

11. Singh S, Sharma S, Basu S. Rabbit models of dry eye disease: Current understanding and unmet needs for translational research. Exp Eye Res. 2021; 206. doi:10.1016/j.exer.2021.108538

12. Sullivan BD, Crews LA, Barıs¸barıs¸sönmez B, et al. Clinical Utility of Objective Tests for Dry Eye Disease: Variability Over Time and Implications for Clinical Trials and Disease Management.; 2012. http://www.accessdata.fda.gov/cdrh_docs/pdf8/K083184.pdf

13. Resau JH, Sakamoto K, Cottrell JR, Hudson EA, Meltzer SJ. Explant Organ Culture: A Review. Vol 7. Kluwer Academic Publishers; 1991.

14. Hutson MS, Alexander PG, Allwardt V, et al. Organs-on-Chips as Bridges for Predictive Toxicology. Appl In Vitro Toxicol. 2016; 2(2): 97-102. doi:10.1089/aivt.2016.0003

15. Esch EW, Bahinski A, Huh D. Organs-on-chips at the frontiers of drug discovery. Nat Rev Drug Discov. 2015; 14(4): 248-260. doi:10.1038/nrd4539

16. Rouen PA, White ML. DRY EYE Prevalence, Assessment, and Management. Vol 36.; 2018. doi:10.1097/NHH.0000000000000652

17. Pflugfelder SC, de Paiva CS. The Pathophysiology of Dry Eye Disease: What We Know and Future Directions for Research. Ophthalmology. 2017; 124(11): S4-S13. doi:10.1016/j.ophtha.2017.07.010

18. Messmer EM. The Pathophysiology, Diagnosis, and Treatment of Dry Eye Disease. Dtsch Arztebl Int. Published online January 30, 2015. doi:10.3238/arztebl.2015.0071

19. Manafi N, Shokri F, Achberger K, et al. Organoids and organ chips in ophthalmology. Ocular Surface. 2021; 19: 1-15. doi:10.1016/j.jtos.2020.11.004

20. Seo J, Huh D. A HUMAN BLINKING ‘EYE-ON-A-CHIP.’ In: 18th International Conference on Miniaturized Systems for Chemistry and Life Sciences. CBM Society; 2014.

21. Bennet D, Estlack Z, Reid T, Kim J. A microengineered human corneal epithelium-on-a-chip for eye drops mass transport evaluation. Lab Chip. 2018; 18(11): 1539-1551. doi:10.1039/c8lc00158h

22. Evinger C, Bao J Bin, Powers AS, et al. Dry eye, blinking, and blepharospasm. Movement Disorders. 2002; 17(SUPPL. 2). doi:10.1002/mds.10065

23. Seo J, Byun WY, Alisafaei F, et al. Multiscale reverse engineering of the human ocular surface. Nat Med. 2019; 25(8): 1310-1318. doi:10.1038/s41591-019-0531-2

24. Heidari M, Noorizadeh F, Wu K, Inomata T, Mashaghi A. Dry eye disease: Emerging approaches to disease analysis and therapy. J Clin Med. 2019; 8(9). doi:10.3390/jcm8091439

25. Seo J, Byun WY, Alisafaei F, et al. Multiscale reverse engineering of the human ocular surface. Nature Medicine. 2019; 25(8): 1310-1318. doi:10.1038/s41591-019-0531-2

26. Selvam S, Thomas PB, Trousdale MD, et al. Tissue-engineered tear secretory system: Functional lacrimal gland acinar cells cultured on matrix protein-coated substrata. J Biomed Mater Res B Appl Biomater. 2007; 80(1): 192-200. doi:10.1002/jbm.b.30584

27. Richards SM, Jensen R V., Liu M, et al. Influence of sex on gene expression in the mouse lacrimal gland. Exp Eye Res. 2006; 82(1): 13-23. doi:10.1016/j.exer.2005.04.014

28. Spaniol K, Metzger M, Roth M, et al. Engineering of a secretory active three-dimensional lacrimal gland construct on the basis of decellularized lacrimal gland tissue. Tissue Eng Part A. 2015; 21(19-20): 2605-2617. doi:10.1089/ten.tea.2014.0694

29. Singh D, Mathur A, Arora S, Roy S, Mahindroo N. Journey of organ on a chip technology and its role in future healthcare scenario. Applied Surface Science Advances. 2022; 9. doi:10.1016/j.apsadv.2022.100246

30. Piergiovanni M, Leite SB, Corvi R, Whelan M. Standardisation needs for organ on chip devices. Lab Chip. 2021; 21(15): 2857-2868. doi:10.1039/d1lc00241d

Received on 15.05.2024 Revised on 27.07.2024

Accepted on 02.09.2024 Published on 11.12.2024

Available online on December 31, 2024

International Journal of Technology. 2024; 14(2):125-131.

DOI: 10.52711/2231-3915.2024.00018

This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License. Creative Commons License.